Fibrinogen, the main structural protein in the blood responsible for the formation of blood clots, exists as a dimer of three polypeptide chains; the Aα (66.5 kD), Bβ (52 kD) and γ (46.5 kD) chains are linked through 29 disulphide bonds. The addition of asparagine-linked carbohydrates to the Bβ and γ chains results in a molecule with a molecular weight of 340 kD. Fibrinogen has a trinodal structure. A central nodule, termed the E domain, contains the amino-termini of all 6 chains including the fibrinopeptides (Fp) whereas the two distal nodules termed D domains contain the carboxy-termini of the Aα, Bβ and γ chains. Fibrinogen is proteolytically cleaved at the amino terminus of the Aα and Bβ chains releasing fibrinopeptides A and B (FpA & FpB) and converted into a fibrin monomer by thrombin, which is a serine protease that is converted from its inactive form by Factor Xa. The resultant fibrin monomers non-covalently assemble into protofibrils by DE contacts on neighboring fibrin molecules, and as a result clots are formed. Patients with a fibrinogen deficiency (either hereditary or acquired) suffer from increased levels of bleeding resembling phenotypes seen in moderate or mild hemophilia.
There has been a need for medicinal fibrinogen to treat increased bleeding or bleeding caused by fibrinogen deficiency. Several sources have been studied and used to produce the molecule, such as purification from human plasma (from donor blood) or through the production of recombinant fibrinogen in prokaryotic cells, eukaryotic cell systems and in the milk of transgenic animals. Although purification of fibrinogen directly from human plasma appeared to be useful, it is accompanied by several potentially severe problems due to the possible presence of human blood-related pathogens such as HIV and others. In addition plasma derived fibrinogen is not consistent in its clotting and cohesion kinetics. The production of recombinant fibrinogen is also hampered by the fact that the protein structure is rather complex as outlined above. The molecule is heavily glycosylated, and it appeared that this glycosylation is of key importance in the functionality of the protein, which makes production on prokaryotes ineffective. The production of fibrinogen in a culture of eukaryotic cells generally resulted in very low yields, which was an indication that such systems could also not provide enough protein for therapeutic use. The solution to these production problems was found in the use of transgenic animals (such as lactating cows) that appeared to be able to produce high concentrations of functional fibrinogen in their milk (WO 95/23868; WO 95/22249).
Although production levels in transgenic mammalian animals are generally high, these animals often experience precipitation of the fibrinogen in the milk (reported as ‘flakes’ and ‘clots’), most likely due to activation of the protein within the mammary gland. This hampers lactation as well as purification of the active molecule. Historically it has been documented that cattle, mice and sheep transgenic for recombinant human fibrinogen (also herein referred to as rhFIb) and expressing it in their milk, often had clots and flakes in their milk. The degree of flakes and clots appears to vary per animal, by expression level, by quarter, and by stage of lactation. Moreover, the degree of aggregation (clotting) can vary on a daily basis and is problematic to control. As far as the inventors are aware, clotting of the milk in animals only occurs upon mastitis, an inflammation of the quarters in the udder.
Clearly, as indicated, such clots trouble the release of milk from the transgenic female animal itself. Moreover, because such aggregation of the transgenically produced fibrinogen hampers the downstream purification process and—through this—lowers the final production level, there is a strong need for compounds and methods and that could prevent this appearance of fibrinogen aggregation and the occurrence of fibrinogen flakes and clots in the milk of animals that are transgenic for rhFib.